Modified alginates for cell encapsulation and cell therapy

ABSTRACT

Covalently modified alginate polymers, possessing enhanced biocompatibility and tailored physiochemical properties, as well as methods of making and use thereof, are disclosed herein. The covalently modified alginates are useful as a matrix for the encapsulation and transplantation of cells. Also disclosed are high throughput methods for the characterizing the biocompatibility and physiochemical properties of modified alginate polymers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority and benefit to U.S. ProvisionalApplication No. 61/492,705, filed Jun. 2, 2011, by Arturo J. Vegas,Kaitlin M. Bratlie, Daniel G. Anderson, and Robert S. Langer.

FIELD OF THE INVENTION

The present invention relates to the use of alginates, chemicallymodified to enhance their biocompatibility and tailor their physicalproperties, for the encapsulation of cells, particularly for theencapsulation of pancreatic islet cells, as well as methods of treatingdiseases or disorders, including diabetes, by implantation of theencapsulated cells.

BACKGROUND OF THE INVENTION

The transplantation of hormone- or protein-secreting cells fromgenetically non-identical members of the same species (i.e.allotransplantation) or from other species (i.e. xenotransplantion) is apromising strategy for the treatment of many diseases and disorders.Using alginate microcapsules to provide immunoisolation, hormone- orprotein-secreting cells can be transplanted into a patient without theneed for extensive treatment with immunosuppressant drugs. Thisprinciple has been successfully demonstrated by the transplantation ofalginate-encapsulated pancreatic β-cells in diabetic rat models (Lim, F.and Sun, A. M. Science. 210, 908-910 (1980)). Methods of encapsulatingbiological material in alginate gels are described, for example, in U.S.Pat. No. 4,352,883 to Lim. In the Lim process, an aqueous solutioncontaining the biological materials to be encapsulated is suspended in asolution of a water soluble polymer. The suspension is formed intodroplets which are configured into discrete microcapsules by contactwith multivalent cations such as Ca²⁺. The surface of the microcapsulesis subsequently crosslinked with polyamino acids, forming asemipermeable membrane around the encapsulated materials.

The Lim method employs conditions which are mild enough to encapsulatecells without adversely affecting their subsequent survival andfunction. The resulting alginate microcapsules are semipermeable,possessing sufficient porosity to permit nutrients, waste, and thehormones and/or proteins secreted from encapsulated cells to diffusefreely into and out of the microcapsules, and, when implanted into ananimal host, the alginate microcapsules effectively isolate theencapsulated cells from the host's immune system. See also U.S. Pat. No.7,807,150 to Vacanti, et al.

Many other synthetic materials have been tried, including blockcopolymers such as polyethyleneglycol-diacrylate polymers,polyacrylates, and thermoplastic polymers, as reported by U.S. Pat. No.6,129,761 to Hubbell and by Aebischer, et al, J Biomech Eng. 1991 May;113(2):178-83. See Lesney Modern Drug Discovery 4(3), 45-46, 49, 50(2001) for review of these materials.

Since Lim first reported on the transplantation of encapsulated cells,many other have tried to create “bioreactors” for cells that couldmaintain viability of the cells in the absence of vascularization, bydiffusion of nutrients, gases and wastes through the encapsulatingmaterials, and still protect the cells from the body's immune defensesagainst foreign cells and materials. Unfortunately, efforts to translatethese therapies into human subjects have proven difficult. For example,alginate-encapsulated porcine islet cells transplanted into a humansubject suffering from Type 1 diabetes initially demonstratedsignificant improvement and required decreased insulin dosing. However,by week 49, the patient's insulin dose retuned to pre-transplant levels(Elliot, R. B. et al. Xenotransplantation. 2007; 14(2): 157-161).

In some cases, it is desirable to elicit fibrosis, for example, when thecells are implanted as a bulking material, as described in U.S. Pat. No.6,060,053 and as subsequently approved by the Food and DrugAdministration for treatment of vesicoureteral reflux.

The diminished efficacy of the implanted cells over time is the resultof fibroblastic overgrowth of the alginate capsules. The alginate gelmatrix provokes an inflammatory response upon implantation, resulting inthe encapsulation of the alginate matrix with fibrous tissue. Thefibrous tissue on the alginate capsule surface reduces the diffusion ofnutrients and oxygen to the encapsulated cells, causing them to die. Nobetter results have been obtained with the other materials.

Therefore, it is an object of the invention to provide polymers suitablefor encapsulation and implantation of cells where the polymers havegreater long term biocompatibility following implantation.

It is another object of the present invention to provide chemicallymodified, ionically crosslinkable alginates with improvedbiocompatibility and tailored physiochemical properties, including gelstability, pore size, and hydrophobicity/hydrophilicity.

It is also an object of the invention to provide methods for theencapsulation of cells using modified alginate polymers.

It is a further object of the invention to provide methods for treatinga disorder or disease in a human or animal patient by transplantingexogenous biological material encapsulated in a modified alginatepolymer.

Finally, it is an object of the invention to provide high-throughputmethods for the characterization of modified alginate polymers.

SUMMARY OF THE INVENTION

Alginates, chemically modified to tailor their biocompatibility andphysical properties, have been developed. The modified alginatesdescribed herein provide enhanced properties relative to unmodifiedalginates. Moreover, based on the discovery that the starting materials,as well as chemically modified and reacted materials, must beexhaustively purified to remove contaminants prior to implantation toprevent encapsulation, these materials are less likely to elicit fibrouscapsule formation following implantation.

Modified alginates are alginate polymers that contain one or morecovalently modified monomers defined by Formula I

wherein,

X is oxygen, sulfur, or NR;

R₁ is hydrogen, or an organic grouping containing any number of carbonatoms, preferably 1-30 carbon atoms, more preferably 1-20 carbon atoms,more preferably 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative R₁ groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

Y₁ and Y₂ independently are hydrogen or —PO(OR)₂; or

Y₂ is absent, and Y₁, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown below

wherein n is an integer between 1 and 4; and

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R groupings being alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R is, independently for each occurrence, hydrogen or an organic groupingcontaining any number of carbon atoms, preferably 1-30 carbon atoms,more preferably 1-20 carbon atoms, more preferably 1-14 carbon atoms,and optionally including one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats,representative R groupings being alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

Modified alginates can be either singularly modified alginate polymersor multiply modified alginate polymers. Singularly modified alginatepolymers are alginate polymers that contain one or more covalentlymodified monomers, wherein substantially all of the covalently modifiedmonomers possess the same covalent modification (i.e. the polymercontains one ‘type’ or species of covalently modified monomer). Multiplymodified alginate polymers are alginate polymers that contain covalentlymodified monomers, wherein substantially all of the covalently modifiedmonomers do not possess the same covalent modification (i.e. the polymercontains two or more ‘types’ or species of covalently modifiedmonomers).

In some embodiments, the modified alginate polymer is a singularlymodified alginate polymer. In preferred embodiments, the modifiedalginate polymer is a multiply modified alginate polymer possessing apolysaccharide backbone containing mannuronate monomers, guluronatemonomers, a first species or type of covalently modified monomer definedby Formula I, and a second species or type of covalently modifiedmonomer defined by Formula I. In some embodiments, the modified alginatepolymer is one of the multiply modified alginate polymers shown below.

Modified alginate polymers can contain any ratio of mannuronatemonomers, guluronate monomers, and covalently modified monomers. Inpreferred embodiments, greater than 20%, more preferably greater than25%, and most preferably greater than 30%, of the monomers in themodified alginate polymer are covalently modified monomers.

In preferred embodiments, the modified alginate polymer can be conicallycrosslinked to form hydrogels using a polyvalent ion, such as Ca²⁺,Sr²⁺, or Ba²⁺. The ability of modified alginates to form stablehydrogels in physiological conditions can be quantified using thehydrogel formation assay described herein. In preferred embodiments, themodified alginate polymer forms hydrogels such that the fluorescenceintensity measured using the high throughput assay described herein isbetween 15,000 and 55,000, preferably between 20,000 and 55,000, morepreferably between 25,000 and 55,000.

In preferred embodiments, the modified alginate is biocompatible, andinduces a lower foreign body response than unmodified alginate. Thebiocompatibility of modified alginates can be quantitatively determinedusing in vitro and in vivo assays known in the field, including the invivo biocompatibility assay described herein. In preferred embodiments,the modified alginate polymer is biocompatible such that thefluorescence response normalized to unmodified alginate measured usingthe in vivo biocompatibility assay described herein is less than 75%,70%, 65%, 60%, 55%, or 50%. Also described are assays for thecharacterization of modified alginate polymers.

A high throughput assay useful to characterize the ability of modifiedalginate polymers to form hydrogels is also described. In someembodiments, the hydrogel formation assay described herein is used toquantify the stability of hydrogels formed from alginates or modifiedalginates. In preferred embodiments, the hydrogel formation assaydescribed herein is used as a screening tool to identify modifiedalginates capable of forming stable hydrogels. The high throughput invivo biocompatibility assay described herein is used to identifymodified alginates which induce a lower foreign body response thanunmodified alginate. Assays are also provided for quantifying thebiocompatibility of modified alginates.

Further described herein are methods of encapsulating biologicalmaterials using modified alginate polymers. In particular embodiments,the modified alginate polymers described herein are used to encapsulatecells for use in methods of treating a disease or disorder in a human oranimal patient. In some embodiments, a disease or disorder in a human oranimal patient is treated by transplanting exogenous biological materialencapsulated in a modified alginate polymer. In particular embodiments,a disease or disorder in a human or animal patient is treated bytransplanting cells encapsulated in a modified alginate polymer. In amore particular embodiment, diabetes is treated by transplantingpancreatic islet cells encapsulated in a modified alginate polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general structure of the modified alginates obtainedusing the combinatorial synthetic approach described in Example 1. Thenumber of alginates prepared with each general structure is indicatedbelow.

FIG. 2 is a plot obtained from the hydrogel formation assay described inExample 2. The average fluorescence intensity values measured formodified alginates are plotted. Modified alginates yielding fluorescencevalues below 15,000 were considered unusable for applications wherehydrogel formation is critical (i.e. the encapsulation of cells).

FIG. 3 is a plot showing the effect of selected modified alginates onHeLa cell line viability as compared to the positive control (noalginate). Alginate (Alg) has a viability of 53%. Several polymers areshown to be more cytotoxic than Alg, however, the majority of thelibrary performs as well or better than Alg.

FIG. 4 is a plot obtained using the in vivo method described in Example5, which quantifies the biocompatibility of selected modified alginates.The fluorescence response obtained for the modified alginates using thein vivo method described in Example 5 was normalized to the fluorescenceresponse measured using unmodified alginate in order to quantify thebiocompatibility of the modified alginates in terms of % fluorescenceresponse.

FIG. 5 is a plot detailing the blood glucose level of mice transplantedwith rat islets encapsulated in selected modified alginates as well astwo different unmodified alginates (CMIT and CJOS). The dashed blackline represents normoglycemia in mice.

FIG. 6 is a bar graph showing inflammatory response (as measured byfluorescence normalized to VLVG) as a function of modified alginate(combined with unmodified alginate).

DETAILED DESCRIPTION OF THE INVENTION

Alginates are a class of linear polysaccharide copolymers formed from1-4-glycosidically linked β-D-mannuronate (M) and its C-5 epimerα-L-guluronate (G). Alginates are naturally occurring biopolymersproduced by a variety of organisms, including marine brown algae and atleast two genera of bacteria (Pseudomonas and Azotobacter). Typically,commercial alginates are isolated from marine algae, includingMacrocystis pyrifera, Ascophyllum nodosum, and various types ofLaminaria.

Three types of primary structure define the polysaccharide backbone ofalginates: homopolymeric regions of consecutive guluronate monomers(G-blocks), homopolymeric regions of consecutive mannuronate monomers(M-blocks), and regions containing alternating mannuronate andguluronate monomers (MG-blocks). The monomer blocks possess differentconformations in solution, ranging from a flexible extended structure(M-blocks) to a rigid compact structure (G-blocks). In the case ofG-blocks, the compact conformation facilitates the chelation ofmultivalent ions, notably Ca²⁺ ions, such that G-blocks in one alginatechain can be ionically crosslinked with G-blocks in another alginatechain, forming stable gels. As a result, the proportion, length, anddistribution of the monomer blocks influence the physiochemicalproperties of the alginate polymer.

In the case of commercially produced alginates obtained from algae, themolecular weight, primary structure, and overall molar ratio of uronicacid monomers (M/G ratio) in the alginate polymer depends on a number offactors, including the species producing the alginate, the time of yearin which the species is collected, and the location and age of the algalbody. As a result, alginates possessing a range of physiochemicalproperties, such as molecular weight and viscosity, are commerciallyavailable.

Alginates can be ionically crosslinked at room temperature and neutralpH to form hydrogels. The ability of alginates to form stable gels inphysiologically compatible conditions renders alginate gels useful in anumber of biomedical applications. For example, alginate gels have beused as a matrix for drug delivery to modulate the pharmacokinetics oftherapeutic, diagnostic, and prophylactic agents.

I. Definitions

“Alginate”, as used herein, is a collective term used to refer to linearpolysaccharides formed from β-D-mannuronate and α-L-guluronate in anyM/G ratio, as well as salts and derivatives thereof. The term“alginate”, as used herein, encompasses any polymer having the structureshown below, as well as salts thereof.

“Biocompatible”, as used herein, refers to a material which performs itsdesired function when introduced into an organism without inducingsignificant inflammatory response, immunogenicity, or cytotoxicity tonative cells, tissues, or organs. Biocompatibility, as used herein, canbe quantified using the in vivo biocompatibility assay described hereinin Example 5.

“Foreign Body Response”, as used herein, refers to the immunologicalresponse of biological tissue to the presence of any foreign material inthe tissue which can include protein adsorption, macrophages,multinucleated foreign body giant cells, fibroblasts, and angiogenesis.

“Chemically Modified Alginate” or “Modified Alginate”, are used hereininterchangeably, and refer to alginate polymers which contain one ormore covalently modified monomers.

“Covalently Modified Monomer”, as used herein, refers to a monomer whichis an analog or derivative of a mannuronate and/or guluronate monomerobtained from a mannuronate and/or guluronate monomer via a chemicalprocess.

“Singularly Modified Alginate Polymer”, as used herein, refers tomodified alginates that contain one or more covalently modifiedmonomers, wherein substantially all of the covalently modified monomerspossess the same covalent modification (i.e. the polymer contains one‘type’ or species of covalently modified monomer). Singularly modifiedalginate polymers include, for example, modified alginate polymerswherein substantially all of the monomers in the modified alginatepolymer are represented by mannuronate monomers, guluronate monomers,and a covalently modified monomer defined by Formula I. Not all of themonomers are necessarily covalently modified.

For clarity of discussion herein, singularly modified alginates aredefined using formulae illustrating the structure of the covalentlymodified monomers incorporated in the backbone and omitting themannuronate and guluronate monomers. For example, a singularly modifiedalginate polymer composed of mannuronate monomers, guluronate monomers,and a covalently modified monomer defined by Formula I, wherein X is NR,R₁ is methyl, and R, Y₁, and Y₂ are hydrogen, is illustrated herein bythe structure below.

“Multiply Modified Alginate Polymer”, as used herein, refers to modifiedalginates that contain covalently modified monomers, whereinsubstantially all of the covalently modified monomers do not possess thesame covalent modification (i.e. the polymer contains two or moredifferent ‘types’ or species of covalently modified monomers). Multiplymodified alginate polymers include, for example, modified alginatepolymers wherein substantially all of the monomers in the modifiedalginate polymer are represented by mannuronate monomers, guluronatemonomers, and two or more different types of covalently modifiedmonomers defined by Formula I. As used in this context, a ‘type’ or‘species’ of covalently modified monomer refers to a covalent monomerdefined by Formula I, wherein all possible variable positions arechemically defined. Not all the monomers are covalently modified.

For clarity of discussion herein, modified alginates are defined usingformulae illustrating the covalently modified monomers incorporated inthe backbone and omitting the mannuronate and guluronate monomers. Forexample, a multiply modified alginate polymer composed of mannuronatemonomers, guluronate monomers, and two different types of covalentlymodified monomers, wherein the first type of covalently modified monomeris defined by Formula I, wherein X is NR, R₁ is methyl, and R, Y₁, andY₂ are hydrogen and the second type of covalently modified monomer isdefined by Formula I, wherein X is oxygen, R₁ is ethyl, and Y₁ and Y₂are hydrogen, is illustrated by the structure below.

“Analog” and “Derivative” are used herein interchangeably, and refer toa compound having a structure similar to that of a parent compound, butvarying from the parent compound by a difference in one or more certaincomponents. Analogs or derivatives differ from the parent compound inone or more atoms, functional groups, or substructures, which arereplaced with other atoms, groups, or substructures. An analog orderivative can be imagined to be formed, at least theoretically, fromthe parent compound via some chemical or physical process. The termsanalog and derivative encompass compounds which retain the same basicring structure as the parent compound, but possess one or more differentsubstituents on the ring(s). For example, analog or derivative ofmannuronate or guluronate refers to compounds which retain the core ofthe monomer, e.g., the pyranose ring, but differ in or moresubstitutents on the ring.

“Mannuronate” and “Mannuronate Monomer”, as used herein, refers tomannuronic acid monomers as well as salts thereof.

“Guluronate” and “Guluronate Monomer”, as used herein, refers toguluronic acid monomers as well as salts thereof.

“Substantially”, as used herein, specifies an amount of 95% or more, 96%or more, 97% or more, 98% or more, or 99% or more.

“Glass Transition Temperature” (T_(g)), as used herein, refers to thetemperature at which a reversible transition is observed in amorphousmaterials from a hard and relatively brittle state into a molten orrubber-like state. Tg values for alginate polymers can be experimentallydetermined using differential scanning calorimetry (DSC, heated andcooled at a rate of 10 K/min). In all cases herein, values of T_(g) aremeasured using powder polymer samples.

“Click Chemistry”, as used herein, refers to chemical reactions used tocouple two compounds together which are high yielding, wide in scope,create only byproducts that can be removed without chromatography, arestereospecific, simple to perform, and can be conducted in easilyremovable or benign solvents. Examples of reactions which fulfill thesecriteria include the nucleophilic ring opening of epoxides andaziridines, non-aldol type carbonyl reactions, including the formationof hydrazones and heterocycles, additions to carbon-carbon multiplebonds, including Michael Additions, and cycloaddition reactions, such asa 1,3-dipolar cycloaddition reaction (i.e. a Huisgen cycloadditionreaction). See, for example, Moses, J. E. and Moorhouse, A. D. Chem Soc.Rev. 2007; 36: 1249-1262; Kolb, H. C. and Sharpless, K. B. DrugDiscovery Today. 2003; 8(24: 1128-1137; and Kolb, H. C., et al. Angew.Chem. Int. Ed. 2001; 40: 2004-2021.

“Polyvalent Cation”, as used herein, refers to cations which have apositive charge greater than 1. Examples include, but are not limitedto, Ca²⁺, Ba²⁺, and Sr²⁺.

“Substituted”, as used herein, refers to all permissible substituents ofthe compounds or functional groups described herein. In the broadestsense, the permissible substituents include acyclic and cyclic, branchedand unbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, preferably 1-14 carbonatoms, and optionally include one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats. Representative substituents include alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, andpolypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e. a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

“Aryl” further encompasses polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

“Alkyl”, as used herein, refers to the radical of saturated orunsaturated aliphatic groups, including straight-chain alkyl, alkenyl,or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups,cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkylsubstituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, andcycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unlessotherwise indicated, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain,C₃-C₃₀ for branched chain), preferably 20 or fewer, more preferably 10or fewer, most preferably 6 or fewer. If the alkyl is unsaturated, thealkyl chain generally has from 2-30 carbons in the chain, preferablyfrom 2-20 carbons in the chain, more preferably from 2-10 carbons in thechain. Likewise, preferred cycloalkyls have from 3-20 carbon atoms intheir ring structure, preferably from 3-10 carbons atoms in their ringstructure, most preferably 5, 6 or 7 carbons in the ring structure.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

“Alkyl” includes one or more substitutions at one or more carbon atomsof the hydrocarbon radical as well as heteroalkyls. Suitablesubstituents include, but are not limited to, halogens, such asfluorine, chlorine, bromine, or iodine; hydroxyl; —NR₁R₂, wherein R₁ andR₂ are independently hydrogen, alkyl, or aryl, and wherein the nitrogenatom is optionally quaternized; —SR, wherein R is hydrogen, alkyl, oraryl; —CN; —NO₂; —COOH; carboxylate; —COR, —COOR, or —CONR₂, wherein Ris hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino,phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃; —CN;—NCOCOCH₂CH₂; —NCOCOCHCH; —NCS; and combinations thereof.

“Amino” and “Amine”, as used herein, are art-recognized and refer toboth substituted and unsubstituted amines, e.g., a moiety that can berepresented by the general formula:

wherein, R, R′, and R″ each independently represent a hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbonyl, —(CH₂)_(m)—R′″, or R and R′ taken together withthe N atom to which they are attached complete a heterocycle having from3 to 14 atoms in the ring structure; R′″ represents a hydroxy group,substituted or unsubstituted carbonyl group, an aryl, a cycloalkyl ring,a cycloalkenyl ring, a heterocycle, or a polycycle; and m is zero or aninteger ranging from 1 to 8. In preferred embodiments, only one of R andR′ can be a carbonyl, e.g., R and R′ together with the nitrogen do notform an imide. In preferred embodiments, R and R′ (and optionally R″)each independently represent a hydrogen atom, substituted orunsubstituted alkyl, a substituted or unsubstituted alkenyl, or—(CH₂)_(m)—R′″. Thus, the term ‘alkylamine’ as used herein refers to anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto (i.e. at least one of R, R′, or R″ is an alkylgroup).

“Carbonyl”, as used herein, is art-recognized and includes such moietiesas can be represented by the general formula:

wherein X is a bond, or represents an oxygen or a sulfur, and Rrepresents a hydrogen, a substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,—(CH₂)_(m)—R″, or a pharmaceutical acceptable salt, R′ represents ahydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, or—(CH₂)_(m)—R″; R″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8. Where X is oxygen and R is defines as above, the moiety isalso referred to as a carboxyl group. When X is oxygen and R ishydrogen, the formula represents a ‘carboxylic acid’. Where X is oxygenand R′ is hydrogen, the formula represents a ‘formate’. In general,where the oxygen atom of the above formula is replaced by a sulfur, theformula represents a ‘thiocarbonyl’ group. Where X is sulfur and R or R′is not hydrogen, the formula represents a ‘thioester’. Where X is sulfurand R is hydrogen, the formula represents a ‘thiocarboxylic acid’. WhereX is sulfur and R′ is hydrogen, the formula represents a ‘thioformate’.Where X is a bond and R is not hydrogen, the above formula represents a‘ketone’. Where X is a bond and R is hydrogen, the above formularepresents an ‘aldehyde’.

“Heteroalkyl”, as used herein, refers to straight or branched chain, orcyclic carbon-containing radicals, or combinations thereof, containingat least one heteroatom. Suitable heteroatoms include, but are notlimited to, O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized.

Examples of saturated hydrocarbon radicals include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, andhomologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, and3-butynyl.

“Alkoxy”, “alkylamino”, and “alkylthio” are used herein in theirconventional sense, and refer to those alkyl groups attached to theremainder of the molecule via an oxygen atom, an amino group, or asulfur atom, respectively.

“Alkylaryl”, as used herein, refers to an alkyl group substituted withan aryl group (e.g., an aromatic or hetero aromatic group).

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocycle orbicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ringatoms, consisting of carbon and one to four heteroatoms each selectedfrom the group consisting of non-peroxide oxygen, sulfur, and N(Y)wherein Y is absent or is H, O, C₁-C₁₀ alkyl, phenyl or benzyl, andoptionally containing 1-3 double bonds and optionally substituted withone or more substituents. Examples of heterocyclic ring include, but arenot limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, (uranyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Heterocyclicgroups can optionally be substituted with one or more substituents asdefined above for alkyl and aryl.

“Halogen”, as used herein, refers to fluorine, chlorine, bromine, oriodine.

II. Modified Alginates

Described herein are alginate polymers that have been chemicallymodified to alter their biocompatibility and physical properties, aswell as methods of making thereof.

A. Structure of Modified Alginate Polymers

Modified alginates contain one or more covalently modified monomersdefined by Formula I

wherein,

X is oxygen, sulfur, or NR;

R₁ is hydrogen, or an organic grouping containing any number of carbonatoms, preferably 1-30 carbon atoms, more preferably 1-20 carbon atoms,more preferably 1-14 carbon atoms, and optionally including one or moreheteroatoms such as oxygen, sulfur, or nitrogen grouping in linear,branched, or cyclic structural formats, representative R₁ groupingsbeing alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, carbonyl, substituted carbonyl, carboxyl, substitutedcarboxyl, amino, substituted amino, amido, substituted amido, sulfonyl,substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl,phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl,C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup;

Y₁ and Y₂ independently are hydrogen or —PO(OR)₂; or

Y₂ is absent, and Y₂, together with the two oxygen atoms to which Y₁ andY₂ are attached form a cyclic structure as shown below

wherein n is an integer between 1 and 4; and

R₂ and R₃ are, independently, hydrogen or an organic grouping containingany number of carbon atoms, preferably 1-30 carbon atoms, morepreferably 1-20 carbon atoms, more preferably 1-14 carbon atoms, andoptionally including one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats,representative R groupings being alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup; or

R₂ and R₃, together with the carbon atom to which they are attached,form a 3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and

R is, independently for each occurrence, hydrogen or an organic groupingcontaining any number of carbon atoms, preferably 1-30 carbon atoms,more preferably 1-20 carbon atoms, more preferably 1-14 carbon atoms,and optionally including one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats,representative R groupings being alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀cyclic, substituted C₃-C₂₀ cyclic, heterocyclic, substitutedheterocyclic, aminoacid, poly(ethylene glycol), peptide, or polypeptidegroup.

In some embodiments, the modified alginate polymer is a singularlymodified alginate polymer. In specific embodiments, the singularlymodified alginate polymer contains one or more covalently modifiedmonomers defined by Formula I, wherein R₁ includes an azide group, analkyne group, or a 1,2,3-triazole ring. In certain embodiments, thesingularly modified alginate polymer contains one or more covalentlymodified monomers defined by Formula I, wherein X is not oxygen and R₁is not an unsubstituted C₁-C₁₈ alkyl group, poly(ethylene glycol) chain,or cholesteryl moiety. In certain additional embodiments, the singularlymodified alginate polymer contains one or more covalently modifiedmonomers defined by Formula I, wherein X is not NR and R₁ is not asubstituted or unsubstituted C₁-C₆ alkyl group, or a poly(ethyleneglycol) chain.

In alternative embodiments, the modified alginate polymer is a multiplymodified alginate polymer. In preferred embodiments, the multiplymodified alginate polymer possesses a polysaccharide backbone containingmannuronate monomers, guluronate monomers, a first species or type ofcovalently modified monomer defined by Formula I, and a second speciesor type of covalently modified monomer defined by Formula I. In otherembodiments, the multiply modified alginate polymer possesses apolysaccharide backbone containing mannuronate monomers, guluronatemonomers, and three or more different types of covalently modifiedmonomers defined by Formula I.

In some embodiments, the multiply modified alginate polymer contains twodifferent species of covalently modified monomers defined by Formula I,wherein in both species of monomer, X is NR. In other embodiments, themultiply modified alginate polymer contains two different species ofcovalently modified monomers defined by Formula I, wherein in bothspecies of monomer, X is oxygen. In further embodiments, the multiplymodified alginate polymer contains two different species of covalentlymodified monomers defined by Formula I, wherein in one species ofmonomer X is oxygen, and in the second species of monomer, X is NR.

In some embodiments, the multiply modified alginate polymer contains twodifferent species of covalently modified monomers defined by Formula I,wherein in at least one species of monomer, R₁ includes one or morecyclic moieties. In preferred embodiments, the multiply modifiedalginate polymer contains two different species of covalently modifiedmonomers defined by Formula I, wherein in at least one species ofmonomer, R₁ includes a phenyl ring, furan ring, oxolane ring, dioxolanering, or a 1,2,3-triazole ring.

In certain embodiments, the multiply modified alginate polymer containstwo different species of covalently modified monomers defined by FormulaI, wherein in at least one species of monomer, R₁ includes one or morehalogen moieties, an azide group, or an alkyne.

In preferred embodiments, the multiply modified alginate polymer is oneof the multiply modified alginate polymers shown below.

Modified alginate polymers can be of any desired molecular weight. Theweight average molecular weight of the alginates is preferably between1,000 and 1,000,000 Daltons, more preferably between 10,000 and 500,000Daltons as determined by gel permeation chromatography.

Modified alginate polymers can contain any ratio of mannuronatemonomers, guluronate monomers, and covalently modified monomers. In someembodiments, greater than 2.5%, 5%, 7.5%, 10%, 12%, 14%, 15%, 16%, 18%,20%, 22%, 24%, 25%, 26%, 28%, 30%, 32.5%, 35%, 37.5%, 40%, 45%, 50%,55%, or 60% of the monomers in the modified alginate polymer arecovalently modified monomers. Preferably greater than 20%, morepreferably greater than 25%, and most preferably greater than 30% of themonomers in the modified alginate polymer are covalently modifiedmonomers.

Modified alginate polymers can be produced incorporating covalentlymodified monomers possessing a range of different hydrogen bondingpotentials, hydrophobicities/hydrophilicities, and charge states. Theinclusion of covalently modified monomers into an alginate polymeralters the physiochemical properties of alginate polymer. Accordingly,the physiochemical properties of alginates can be tuned for desiredapplications by the selective incorporation of covalently modifiedmonomers.

For example, the glass transition temperature (T_(g)), can be varied bythe incorporation of covalently modified monomers. In some embodiments,the modified alginate polymer powder possess a T_(g), as measured bydifferential scanning calorimetry (DSC), of greater than 50° C., 60° C.,65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105°C., 110° C., 115° C., 120° C., 125° C., 130° C., 135° C., 140° C., 145°C., 150° C., 160° C., 175° C., 190° C., or 200° C.

The hydrophobicity/hydrophilicity of alginates can be varied by theincorporation of hydrophobic and/or hydrophilic covalently modifiedmonomers. In preferred embodiments, the modified alginate polymercontains one or more hydrophobic covalently modified monomers. Therelative hydrophobicity/hydrophilicity of modified alginates can bequantitatively assessed by measuring the contact angle of a waterdroplet on a film of the modified alginate polymer using a goniometer.In some embodiments, the modified alginate has a contact angle of lessthan 90° (i.e. it is hydrophilic). In preferred embodiments, themodified alginate has a contact angle of more than 90° (i.e. it ishydrophobic). In some embodiments, the modified alginate has a contactangle of more than 95°, 100°, 105°, 110°, 115°, or 120°.

In embodiments used for cell encapsulation, the modified alginatepolymer can be ionically crosslinked by a polyvalent cation such asCa²⁺, Sr²⁺, or Ba²⁺ to form hydrogels. The ability of modified alginatesto form stable hydrogels in physiological conditions can be quantifiedusing the hydrogel formation assay described in Example 2.

In some embodiments, the modified alginate polymer forms hydrogels suchthat the fluorescence intensity measured using the high throughputhydrogel formation assay described herein is greater than 10,000,15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or55,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is greater than15,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is between 15,000and 55,000, preferably between 20,000 and 55,000, more preferablybetween 25,000 and 55,000.

In embodiments used for cell encapsulation, the modified alginatepolymer forms a hydrogel with sufficient porosity to permit nutrients,waste, and the hormones and/or proteins secreted from encapsulated cellsto diffuse freely into and out of the microcapsules, whilesimultaneously preventing the incursion of immune cells into the gelmatrix. The porosity and surface area of modified alginate hydrogels canbe measured using BET analysis. Prior to BET analysis, solvent andvolatile impurities are removed by prolonged heating of the modifiedalginate gel under vacuum. Subsequently, the hydrogel samples are cooledunder vacuum, for example by liquid nitrogen, and analyzed by measuringthe volume of gas (typically N₂, Kr, CO₂, or Ar gas) adsorbed to thehydrogel at specific pressures. Analysis of the physisorption of the gasat variable pressures is used to characterize the total surface area andporosity of gels formed by the modified alginate polymers. The preferredmethod of determining hydrogel porosity is BET analysis.

In preferred embodiments, the modified alginate forms a hydrogel withsufficient porosity to permit nutrients, waste, and the hormones and/orproteins secreted from encapsulated cells to diffuse freely into and outof the microcapsules, while simultaneously preventing the incursion ofimmune cells into the gel matrix. In some embodiments, the porosity ofthe hydrogel formed by the modified alginate polymer is increased by 5%,10%, 15%, or 20% relative to the porosity of a hydrogel formed from theunmodified alginate polymer. In alternative embodiments, the porosity ofthe hydrogel formed by the modified alginate polymer is decreased by 5%,10%, 15%, or 20% relative to the porosity of a hydrogel formed from theunmodified alginate polymer.

In preferred embodiments used for cell encapsulation, the modifiedalginate is biocompatible. The biocompatibility of modified alginatescan be quantitatively determined using the fluorescence-based in vivobiocompatibility assay described in Example 5. In this assay, cathepsinactivity was measured using an in vivo fluorescence assay to quantifythe foreign body response to the modified alginate.

In some embodiments, the modified alginate polymer is biocompatible suchthat the fluorescence response normalized to unmodified alginatemeasured using the in viva biocompatibility assay described herein isless than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,or 40%. In preferred embodiments, the modified alginate polymer inducesa lower foreign body response than unmodified alginate. This isindicated by fluorescence response normalized to unmodified alginate ofless than 100%. In some embodiments, the modified alginate polymer isbiocompatible such that the fluorescence response normalized tounmodified alginate measured using the in vivo biocompatibility assaydescribed herein is less than 75%, more preferably less than 65%, andmost preferably less than 50%.

B. Particle Morphology

The growing recognition of the parameters driving fibrosis in vivo hasbeen applied to the analysis of the performance of modified alginates.Intraperitoneal (IP) implantation of modified alginate capsules revealedthat modified alginates may result in abnormally shaped capsules whencrosslinked using conditions defined for unmodified alginates. Theseabnormally shaped capsules can complicate implementation andinterpretation of modified alginate capsules implanted IP. In an effortto improve the capsule morphology, formulation methods for use withmodified alginate microparticles were developed where modified alginateswere blended with a small amount of high molecular weight alginate.Particles prepared from this mixture yielded particles with improvedmorphology and stability.

The unmodified alginate typically has a weight average molecular weightof about 50,000 Daltons to about 500,000 Daltons; however, unmodifiedalginates having molecular weights can also be used. In someembodiments, the weight average molecular weight is from about 50,000 toabout 250,000 Daltons, more preferably from about 50,000 to about150,000 Daltons. In some embodiments, the weight average molecularweight is about 100,000 Daltons.

In other embodiments, one or more additional hydrogel-forming polymersare used in combination with unmodified alginate or in place ofunmodified alginate. Such polymers are known in the art. Examplesinclude, but are not limited to, PEG, chitosan, dextran, hyaluronicacid, silk, fibrin, polyvinyl alcohol) and poly(hydroxyl ethylmethacrylate).

For example, particles prepared from modified alginate 263_A12microparticles formulated with barium and mannitol were compared toparticles prepared from 263_A12 blended with a small amount ofunmodified SLG100 alginate (16% by weight). The particles prepared froma mixture of modified alginate and unmodified alginate produced morehomogenous microparticle populations in terms of shape and size asevaluated by scanning electron microscopy (SEM). Quantitativefluorescence analysis with prosense at several time points with modifiedalginates blended with SLG100 showed that several reformulated modifiedalginates display less inflammatory response at day 7 compared to thecontrol alginate. Initial experiments with large capsules (1.5 mmdiameter) were comparably clean capsules after 2 weeks in the IP spaceof immunocompetent C57BL6 mice.

C. Preparation of Modified Alginate Polymers

Modified alginates can be prepared through covalent modification of anyavailable alginate polymer. Covalently modified monomers can beintroduced into alginate polymers using a variety of syntheticprocedures known in the art. In some embodiments, mannuronate andguluronate monomers are covalently modified via esterification and/oramidation of their carboxylic acid moiety. In alternative embodiments,mannuronate and guluronate monomers are covalently modified viaphosphorylation or acetal formation. Stoichiometric variation of thereactants during covalent modification can be used to vary the amount ofcovalently modified monomer incorporated into the modified alginate.

In addition to the reactions discussed below, alternative syntheticmethodologies for the covalent modification of mannuronate andguluronate monomers are known in the art. (see, for example, March,“Advanced Organic Chemistry,” 5^(th) Edition, 2001, Wiley-IntersciencePublication, New York).

1. Modification Via the Carboxylate Moiety of the Mannuronate andGuluronate Monomers

Mannuronate and guluronate monomers contain a carboxylic acid moietywhich can serve as a point of covalent modification. In preferredembodiments, the carboxylic acid moiety present on one or moremannuronate and/or guluronate residues (1) are reacted as shown inScheme 1.

Mannuronate and guluronate residues (A) can be readily esterified by avariety of methods known in the art, forming covalently modified monomerB. For example, using a Steglich Esterification, mannuronate andguluronate residues (A) can be esterified by reaction with any suitablealcohol (HO—R₁) in the presence of a carbodiimide (for example,N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)) anddimethylaminopyridine (DMAP). In a preferred method, mannuronate andguluronate residues (A) were esterified by reaction with a large molarexcess of an alcohol (HO—R₁) in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). See, for example, Garrett, C. E. et al. Tetrahedron Lett. 2002;43(23): 4161-4164. Preferred alcohols for use as reagents inesterification include those shown below.

Mannuronate and guluronate residues (A) can also be covalently modifiedvia amidation, forming modified monomer C. For example, mannuronate andguluronate residues (A) can amidated by reaction with any suitable amine(R₁—NH₂) in the presence of a carbodiimide and DMAP. In a preferredmethod, mannuronate and guluronate residues (A) were amidated byreaction with a stoichiometric amount of a suitable amine (R₁—NH₂) inthe presence of CDMT and NMM. Preferred amines for use as reagents inamidation reactions include those shown below.

2. Modification of Mannuronate and Guluronate Monomers Via ClickChemistry

In some embodiments, mannuronate and guluronate monomers are covalentlymodified to introduce a functional group which can be further reactedvia click chemistry.

In preferred embodiments, amidation and/or esterification is used tointroduce a functional group which can further reacted using a1,3-dipolar cycloaddition reaction (i.e. a Huisgen cycloadditionreaction). In a 1,3-dipolar cycloaddition reaction, a first moleculecontaining an azide moiety is reacted with a second molecule containinga terminal or internal alkyne. As shown below, the azide and the alkynegroups undergo an intramolecular 1,3-dipolar cycloaddition reaction,coupling the two molecules together and forming a 1,2,3-triazole ring.

The regiochemistry of 1,3-dipolar cycloadditions reaction can becontrolled by addition of a copper(I) catalyst (formed in situ by thereduction of CuSO₄ with sodium ascorbate) or a ruthenium catalyst (suchas Cp*RuCl(PPh₃)₂, Cp*Ru(COD), or Cp*[RuCl₄]). For example, using acopper catalyst, azides and terminal alkynes can be reacted toexclusively afford the 1,4-regioisomers of 1,2,3-triazoles. Similarly,in the presence of a suitable ruthenium catalyst, azides can be reactedwith internal or terminal alkynes to form exclusively the1,5-regioisomers of 1,2,3-triazoles.

In some embodiments, amidation and/or esterification is used to form acovalently modified monomer containing an alkyne moiety. In theseembodiments, the alkyne moiety present on the covalently modifiedmonomer can be further reacted with a second molecule containing anazide functional group. Upon reaction, the azide and the alkyne groupsundergo an intramolecular 1,3-dipolar cycloaddition reaction forming a1,2,3-triazole ring, coupling the second molecule to the covalentlymodified monomer.

In alternative embodiments, amidation and/or esterification is used toform a covalently modified monomer containing an azide moiety. In theseembodiments, the azide moiety present on the covalently modified monomercan be further reacted with a second molecule containing a terminal orinternal alkyne. Upon reaction, the azide and the alkyne groups undergoan intramolecular 1,3-dipolar cycloaddition reaction forming a1,2,3-triazole ring, coupling the second molecule to the covalentlymodified monomer.

In preferred embodiments, amidation is used to form a covalentlymodified monomer containing an azide moiety. Subsequently, the azidemoiety present on the covalently modified monomer is reacted with asecond molecule containing a terminal or internal alkyne, forming a1,2,3-triazole ring and coupling the second molecule to the covalentlymodified monomer.

As shown in Scheme 2, different strategies can be employed to preparecovalently modified monomers containing an azide moiety. For example,mannuronate and guluronate residues (A) can amidated by reaction with anamine substituted with an azide moiety (for example,11-Azido-3,6,9-trioxaundecan-1-amine) in the presence of a carbodiimideand DMAP, forming azide-functionalized modified monomer F in a singlesynthetic step. Alternatively, mannuronate and guluronate residues (A)can amidated by reaction with an amine substituted with any moiety whichcan be readily transformed into an azide. For example, mannuronate andguluronate residues can be amidated by reaction with 4-iodobenzylaminein the presence of a carbodiimide and DMAP, forming iodo-functionalizedmonomer D. The iodine moiety can then be readily converted to the azide,for example by treatment with sodium azide.

Subsequently, the azide-functionalized monomers can be reacted with amolecule containing an alkyne functionality. For example,azide-functionalized monomers F and E can be reacted with a moleculecontaining a terminal alkyne functionality in the presence of acopper(I) catalyst (formed in situ by the reduction of CuSO₄ with sodiumascorbate), forming covalently modified monomers G and H.

Preferred alkynes for use as reagents in 1,3-dipolarcycloadditionreactions include those shown below.

3. Modification Via the Hydroxyl Moiety of the Mannuronate andGuluronate Monomers

Mannuronate and guluronate monomers contain hydroxyl moieties which canserve as a point of covalent modification. In preferred embodiments, thehydroxyl moieties of mannuronate and guluronate residues (1) are reactedas shown in Scheme 3.

Representative Reaction Conditions: i. I—PO(OR)₂, pyridine; ii.R₂—CO—R₃, H⁺.

Mannuronate and guluronate residues (A) can be phosphorylated by avariety of methods known in the art, forming covalently modified monomerI. For example, mannuronate and guluronate residues can bephosphorylated by reaction with I—PO(OR)₂ in the presence of pyridine(Stowell, 3. K. and Widlanski, T. S. Tetrahedron Lett. 1995; 36(11):1825-1826.).

Mannuronate and guluronate residues (A) can also be converted to acyclic acetal using procedures known in the art. For example, a cyclicacetal can be formed by reaction of mannuronate and guluronate residueswith any suitable ketone (R₂—CO—R₃) in acidic conditions.

4. Methods for Preparing Multiply Modified Alginate Polymers

In the case of singularly modified alginate polymers, only a singlereaction or sequence of reactions is performed, introducing one type ofcovalently modified monomer.

In the case of multiply modified alginate polymers, one or morereactions are performed to introduce multiple different types ofcovalently modified monomers into the modified alginate polymer. In someembodiments, multiply modified alginate polymers are prepared usingmultiple sequential synthetic reactions. For example, the multiplymodified alginate polymer shown below can be prepared using twosequential reactions: (1) amidation of mannuronate and guluronatemonomers with methylamine in the presence of CDMT and NMM; and (2)esterification of mannuronate and guluronate residues with ethanol inthe presence of CDMT and NMM.

In alternative embodiments, multiply modified alginate polymers can beprepared using a ‘one-pot’ synthesis. In these embodiments, multiplecovalently modified monomers are introduced into the alginate polymer ina single synthetic step. For example, the multiply modified alginatepolymer shown above can alternatively be prepared in a single syntheticstep by reacting an alginate polymer with methylamine and ethanol in thepresence of CDMT and NMM.

C. Purification of Alginates

Commercial alginates are generally obtained from algae. Crude alginatesfrom seaweed contain numerous contaminants, including polyphenols,proteins, and endotoxins (de Vos, P, et al. Biomaterials 2006; 27:5603-5617). The presence of these impurities has been shown to limit thebiocompatibility of implanted alginates.

To optimize the biocompatibility of the chemically modified alginatesdescribed herein, a rigorous purification methodology was developed toeliminate potentially irritating impurities. In preferred embodiments,ultra-pure low viscosity alginate (UPVLVG, FMC Biopolymer) was used as asubstrate for covalent modification. Following each covalentmodification, the modified alginates were filtered through modifiedsilica columns, for example cyano-modified silica columns, aimed atcapturing bulk organic impurities. Finally, after covalent modificationof the alginate polymer is complete, the modified alginates are dialyzedto remove any remaining small-molecule or low molecular weightimpurities. In a preferred method, the modified alginates are dialyzedagainst 10,000 molecular weight cut-off (MWCO) membrane to remove anyremaining small-molecule impurities.

The purity of the modified alginates can be determined by ¹H NMRanalysis. In such an analysis, the ¹H NMR spectra of the modifiedalginate polymer is collected, and peaks corresponding to the modifiedalginate polymer and to any impurities are integrated to determine therelative quantity of each species in the sample. In some embodiments,the purity of the modified alginate polymer, as determined by ¹H NMR, isgreater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Inpreferred embodiments, the purity of the modified alginate polymer, asdetermined by ¹H NMR, is greater than 90%, more preferably greater than95%.

III. Assays for the Characterization of Modified Alginate Polymers

The covalent modification of alginate polymers alters the physiochemicalproperties and biological compatibility of the alginate polymer.

In some embodiments, a hydrogel formation assay is used to quantify thestability of hydrogels formed from alginates or modified alginates. Inpreferred embodiments, the hydrogel formation assay is used as ascreening tool to identify modified alginates capable of forming stablehydrogels.

In vivo assays useful to characterize the biocompatibility of modifiedalginate polymers. In some embodiments, the high throughput in vivobiocompatibility assay described herein is used to identify modifiedalginates which induce a lower foreign body response than unmodifiedalginate.

Further described herein is an in vivo method for quantifying thebiocompatibility of modified alginates.

The assays can be used to assess the suitability and biocompatibility ofboth modified and unmodified alginates for certain applications.

A. High Throughput Hydrogel Formation Assay

Covalent modification of the alginates affects the physical propertiesof the alginate, including the ability of the alginate to form hydrogelssuitable for the encapsulation of cells and biomolecules.

The gel-forming assay exploits the ability of hydrogels to trapfluorescent compounds, and differentially retain the fluorophores uponwashing based on the stability of the hydrogel. In this assay, ahydrogel formed by ionically crosslinking a candidate modified alginatein aqueous solution containing a dissolved fluorophore. A variety offluorophores can be used in this assay. In preferred embodiments, thefluorophores possesses an emission maxima between 480 and 750 nm. Inpreferred embodiments, the fluorophore is a rhodamine dye possessing anemission maximum between 550 and 600 nm.

After crosslinking, the hydrogel is repeatedly washed with water.Candidate modified alginates which do not efficiently crosslink arewashed away along with any fluorophore present. Modified alginates whichefficiently crosslink retain the fluorophore during washes. Accordingly,by measuring the fluorescence of modified alginate hydrogels afterwashing, modified alginates capable of forming stable hydrogels can bereadily identified.

In some embodiments, the relative fluorescence intensity values measuredfor a modified alginate are compared relative to fluorescence levelsmeasured for the negative control and unmodified alginate to determineif the modified alginate is capable of forming hydrogels. In alternativeembodiments, the hydrogel formation assay described herein is used toquantify the stability of hydrogels formed from alginates or modifiedalginates. In these embodiments, the fluorescence intensity measured fora modified alginate is used to indicate the stability of the hydrogelformed by the alginate.

In preferred embodiments, the modified alginate polymer forms hydrogelssuch that the fluorescence intensity measured using the high throughputhydrogel formation assay described herein is greater than 10,000,15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or55,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is greater than15,000. In preferred embodiments, the modified alginate polymer formshydrogels such that the fluorescence intensity measured using the highthroughput hydrogel formation assay described herein is between 15,000and 55,000, more preferably between 25,000 and 55,000.

B. High Throughput In Vivo Biocompatibility Assay

Current biocompatibility analysis methods are slow and requirehistological analysis. Described herein is a high throughput in vivobiocompatibility assay, useful for assessing the relativebiocompatibility of alginate polymers.

In the high throughput in vivo biocompatibility assay described herein,modified alginate polymers and an unmodified alginate control areinjected in an array format on the back of an animal test subject tofacilitate high-throughput screening. In preferred embodiments, theanimal test subject is a mouse. After injection, cathepsin activity atthe point of injection of the modified alginates was compared tocathepsin activity at the point of injection the unmodified alginate tocompare the foreign body response to the implanted alginates using invivo fluorescence imaging. In preferred embodiments, thebiocompatibility of the materials was assessed at 14 days post injectionusing in vivo fluorescence imaging.

In preferred embodiments, the high throughput in vivo biocompatibilityassay described herein is used to identify modified alginates whichinduce a lower foreign body response than unmodified alginate. Thefluorescence intensity measured at the implantation site of modifiedalginates was compared with the fluorescence intensity measured at theimplantation site of an unmodified alginate. In preferred embodiments,modified alginates exhibiting a smaller fluorescence intensity at theimplantation site than the fluorescence intensity measured at theimplantation site of an unmodified alginates were qualitativelycharacterized as biocompatible. Conversely, modified alginatesexhibiting a greater fluorescence intensity at the implantation sitethan the fluorescence intensity measured at the implantation site of anunmodified alginates were characterized as not biocompatible.

The high throughput in vivo biocompatibility assay described above canalso be used to characterize the ability of modified alginates to formmechanically stable hydrogels in vivo. In preferred embodiments, the inviva stability of the alginate gels was assessed at 28 days postinjection.

In preferred embodiments, modified alginates gels which remained at thesite of injection after 28 days were characterized as capable of formingmechanically stable hydrogels in vivo. Conversely, modified alginategels which were not present at the site of injection after 28 days weredeemed to not capable of forming mechanically stable hydrogels in vivo.

C. In Vivo Screening of Modified Alginates to Quantify Biocompatibility

Further described herein is an in viva method for quantifying thebiocompatibility of modified alginates.

In this method, a modified alginate polymers is injected on the back ofan animal test subject. In preferred embodiments, the animal testsubject is a mouse. After injection, cathepsin activity at the point ofinjection of the modified alginates was measured using in vivofluorescence assay. In preferred embodiments, the fluorescence assay wasconducted at 7 days post injection using in vivo fluorescence imaging.In preferred embodiments, the fluorescence intensity was measured andnormalized to the fluorescence response measured using unmodifiedalginate in order to quantify the biocompatibility of the modifiedalginates.

In preferred embodiments, the modified alginate polymer induces a lowerforeign body response than unmodified alginate (i.e. the fluorescenceresponse normalized to unmodified alginate is less that 100%). In someembodiments, the modified alginate polymer is biocompatible such thatthe fluorescence response normalized to unmodified alginate measuredusing the in vivo biocompatibility assay described herein is less than95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%. Inpreferred embodiments, the modified alginate polymer is biocompatiblesuch that the fluorescence response normalized to unmodified alginatemeasured using the in vivo biocompatibility assay described herein isless than 75%, more preferably less than 65%, and most preferably lessthan 50%.

IV. Methods of Use

Alginates are used in a variety of applications in the food,pharmaceutical, cosmetic, agriculture, printing, and textile industries.Alginates are widely employed in the food industry as thickening,gelling, stabilizing, bodying, suspending, and emulsifying agents.Alginates can be used as a matrix to control the delivery oftherapeutic, prophylactic, and/or diagnostic agents. Alginates can beincorporated in pharmaceutical compositions as excipients, where theycan act as viscosifiers, suspension agents, emulsifiers, binders, anddisintigrants. Alginate also used as a dental impression material,component of wound dressings, and as a printing agent. One of ordinaryskill in the art will recognize that the modified alginates disclosedherein can be used in any application for which alginates are currentlyemployed.

It is specifically contemplated that modified alginates described hereincan be used in applications where improved biocompatibility and physicalproperties, as compared to commercially available alginates, arepreferred.

A. Encapsulation of Cells

Alginate can be conically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. See, for example, inU.S. Pat. No. 4,352,883 to Lim. In the Lim process, an aqueous solutioncontaining the biological materials to be encapsulated is suspended in asolution of a water soluble polymer, the suspension is formed intodroplets which are configured into discrete microcapsules by contactwith multivalent cations, then the surface of the microcapsules iscrosslinked with polyamino acids to form a semipermeable membrane aroundthe encapsulated materials.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N+--\/\/\/--+NR₃ can also be used. Aqueoussolutions of the salts of these cations are added to the polymers toform soft, highly swollen hydrogels and membranes. The higher theconcentration of cation, or the higher the valence, the greater thedegree of cross-linking of the polymer. Concentrations from as low as0.005 M have been demonstrated to cross-link the polymer. Higherconcentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of polymers containing basicsidechains to form a hydrogel are divalent and trivalent anions such aslow molecular weight dicarboxylic acids, for example, terephthalic acid,sulfate ions and carbonate ions. Aqueous solutions of the salts of theseanions are added to the polymers to form soft, highly swollen hydrogelsand membranes, as described with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

In a preferred method, cells are encapsulated in a modified alginatepolymer. In a preferred embodiment, modified alginate capsules arefabricated from solution of modified alginate containing suspended cellsusing the encapsulator (such as an Inotech encapsulator). In someembodiments, modified alginate are ionically crosslinked with apolyvalent cation, such as Ca²⁺, Ba²⁺, or Sr²⁺. In particularlypreferred embodiments, the modified alginate is crosslinked using BaCl₂.In some embodiments, the capsules are further purified after formation.In preferred embodiments, the capsules are washed with, for example,HEPES solution, Krebs solution, and/or RPMI-1640 medium.

Cells can be obtained directed from a donor, from cell culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiments, cells are obtained directly from a donor, washed andimplanted directly in combination with the polymeric material. The cellsare cultured using techniques known to those skilled in the art oftissue culture. In the preferred embodiment, the cells areautologous—i.e., derived from the individual into which the cells are tobe transplanted, but may be allogeneic or heterologous.

Cell attachment and viability can be assessed using scanning electronmicroscopy, histology, and quantitative assessment with radioisotopes.The function of the implanted cells can be determined using acombination of the above-techniques and functional assays. For example,in the case of hepatocytes, in vivo liver function studies can beperformed by placing a cannula into the recipient's common bile duct.Bile can then be collected in increments. Bile pigments can be analyzedby high pressure liquid chromatography looking for underivatizedtetrapyrroles or by thin layer chromatography after being converted toazodipyrroles by reaction with diazotized azodipyrrolesethylanthranilate either with or without treatment with P-glucuronidase.Diconjugated and monoconjugated bilirubin can also be determined by thinlayer chromatography after alkalinemethanolysis of conjugated bilepigments. In general, as the number of functioning transplantedhepatocytes increases, the levels of conjugated bilirubin will increase.Simple liver function tests can also be done on blood samples, such asalbumin production. Analogous organ function studies can be conductedusing techniques known to those skilled in the art, as required todetermine the extent of cell function after implantation. For example,islet cells of the pancreas may be delivered in a similar fashion tothat specifically used to implant hepatocytes, to achieve glucoseregulation by appropriate secretion of insulin to cure diabetes. Otherendocrine tissues can also be implanted. Studies using labeled glucoseas well as studies using protein assays can be performed to quantitatecell mass on the polymer scaffolds. These studies of cell mass can thenbe correlated with cell functional studies to determine what theappropriate cell mass is. In the case of chondrocytes, function isdefined as providing appropriate structural support for the surroundingattached tissues.

This technique can be used to provide multiple cell types, includinggenetically altered cells, within a three-dimensional scaffolding forthe efficient transfer of large number of cells and the promotion oftransplant engraftment for the purpose of creating a new tissue ortissue equivalent. It can also be used for immunoprotection of celltransplants while a new tissue or tissue equivalent is growing byexcluding the host immune system.

Examples of cells which can be implanted as described herein includechondrocytes and other cells that form cartilage, osteoblasts and othercells that form bone, muscle cells, fibroblasts, and organ cells. Asused herein, “organ cells” includes hepatocytes, islet cells, cells ofintestinal origin, cells derived from the kidney, and other cells actingprimarily to synthesize and secret, or to metabolize materials. Apreferred cell type is a pancreatic islet cell.

The polymeric matrix can be combined with humoral factors to promotecell transplantation and engraftment. For example, the polymeric matrixcan be combined with angiogenic factors, antibiotics,antiinflammatories, growth factors, compounds which inducedifferentiation, and other factors which are known to those skilled inthe art of cell culture.

For example, humoral factors could be mixed in a slow-release form withthe cell-alginate suspension prior to formation of implant ortransplantation. Alternatively, the hydrogel could be modified to bindhumoral factors or signal recognition sequences prior to combinationwith isolated cell suspension.

The techniques described herein can be used for delivery of manydifferent cell types to achieve different tissue structures. In thepreferred embodiment, the cells are mixed with the hydrogel solution andinjected directly into a site where it is desired to implant the cells,prior to hardening of the hydrogel. However, the matrix may also bemolded and implanted in one or more different areas of the body to suita particular application. This application is particularly relevantwhere a specific structural design is desired or where the area intowhich the cells are to be implanted lacks specific structure or supportto facilitate growth and proliferation of the cells.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. For cellshaving organ function, for example, hepatocytes or islet cells, themixture can be injected into the mesentery, subcutaneous tissue,retroperitoneum, properitoneal space, and intramuscular space. Forformation of cartilage, the cells are injected into the site wherecartilage formation is desired. One could also apply an external mold toshape the injected solution. Additionally, by controlling the rate ofpolymerization, it is possible to mold the cell-hydrogel injectedimplant like one would mold clay. Alternatively, the mixture can beinjected into a mold, the hydrogel allowed to harden, then the materialimplanted.

B. Treatment of Diseases or Disorders

Encapsulated cells can be transplanted into a patient in need thereof totreat a disease or disorder. In some embodiments, the encapsulated cellsare obtained from a genetically non-identical member of the samespecies. In alternative embodiments, the encapsulated cells are obtainedfrom a different species than the patient. In preferred embodiments,hormone- or protein-secreting cells are encapsulated and transplantedinto a patient to treat a disease or disorder.

In preferred embodiments, the disease or disorder is caused by orinvolves the malfunction hormone- or protein-secreting cells in apatient. In a preferred embodiment, the disease or disorder is diabetes.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: Combinatorial Synthesis of Chemically ModifiedAlginates

The determinate parameters governing material biocompatibility arepoorly understood. Accordingly, the rational design and synthesis ofmodified alginates possessing improved biocompatibility is challenging.In an effort to identify modified alginates with improvedbiocompatibility and physical properties, a combinatorial approach wasused to prepare a library of modified alginates possessing a range ofcovalent modifications.

1. General Combinatorial Strategy

A pool of twelve alcohols, nine amines, two amines used to introduce anazide moiety (one amine containing an azide moiety and one aminecontaining an iodide moiety to be converted to an azide moietysubsequent to amidation), and nineteen alkynes with a range of differentchemical structures, hydrophobicities/hydrophilicities, hydrogen-bondingpotentials, and charge states were selected as reagents for thecombinatorial synthesis of modified alginates. With the knowledge thatimpurities present in alginate polymers have been shown to limit thebiocompatibility of implanted alginates, ultra-pure, low viscosityalginate (UPLVG, FMC Biopolymers) was selected as a starting materialfor modification experiments.

Unmodified alginate polymer was covalently modified by reaction withone, two, or three the esters, amines, and/or alkynes shown above in acombinatorial fashion. FIG. 1 shows the general structure of themodified alginates obtained using this method.

2. Representative Reaction Conditions

Due to the parallel and combinatorial nature of the modificationprocess, synthetic reactions were performed using a robotic core module.UPLVG alginate was selected as a starting material for modificationexperiments. In the first combinatorial reaction, the unmodifiedalginate was reacted with one of the alcohols, amines, and amines usedto introduce an azide moiety in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). In a second combinatorial step, each of the modified alignatepolymers formed above was reacted with another of the alcohols, amines,or amines used to introduce an azide moiety in the presence of2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methyl morpholine(NMM). In a final combinatorial step, all members of the library whichwere reacted with an amine used to introduce an azide moiety werefurther functionalized using a 1,3-dipolar cycloaddition reaction. Thosemembers of the library which had been reacted with 4-iodobenzylaminewere first reacted with sodium azide to convert the iodide moieties toazide moieties. Subsequently, all members of the library which werereacted with an amine used to introduce an azide moiety were reactedwith one of the alkynes used as reagents for 1,3-dipolar cycloadditionin the presence of CuSO₄/sodium ascorbate.

To optimize the biocompatibility of the chemically modified alginates, arigorous purification methodology was developed to eliminate potentiallyirritating impurities. Following each covalent modification, themodified alginates were filtered through a cyano-modified silica columnaimed at capturing bulk organic impurities. Finally, after completingall covalent modification steps, the modified alginates were dialyzedagainst 10,000 MWCO membrane to remove any remaining small-molecule orlow molecular weight impurities.

The purity of the modified alginates was determined by ¹H NMR analysis.The ¹H NMR spectra of each modified alginate polymer was collected, andpeaks corresponding to the modified alginate polymer and to anyimpurities were integrated to determine the relative quantity of eachspecies in the sample.

Example 2: High Throughput Screening of Modified Alginates Using aHydrogel Formation Assay

Covalent modification of the alginates affects the physical propertiesof the alginate, including the ability of the alginate to form hydrogelssuitable for the encapsulation of cells and biomolecules. To eliminatemodified alginates that have lost their ability to form hydrogels and tofurther focus our screening efforts on stronger candidates, afluorescence-based crosslinking assay was used to quantify the abilityof modified alginates to form hydrogels.

The hydrogel formation assay described herein exploits the ability ofhydrogels to trap fluorescent compounds, and differentially retain thefluorophores upon washing based on the stability of the hydrogel. Eachof the modified alginates prepared using the combinatorial approachdescribed in Example 1 was dissolved in water. A rhodamine dye thatfluoresces at 580 nm was added to each sample. The modified alginatesample was then crosslinked by the addition of a barium or calcium salt,for example BaCl₂, to induce formation of a hydrogel. After incubationfor 10 minutes, each sample was washed repeatedly with water. Thefluorescence intensity of each sample was measured using a fluorimeter.

Each modified alginate was screened three times, and the resultsobtained in the three trials were averaged. The average fluorescenceintensity values for each modified alginate were compared to thefluorescence levels of the negative control (water) and unmodifiedalginate (UPLVG). Modified alginates yielding fluorescence values belowthe negative control were considered unusable for applications wherehydrogel formation is critical (i.e. the encapsulation of cells).

Example 3: In Vitro Screening of Modified Alginates for Biocompatibility

The cytotoxicity of the modified alginate polymers on HeLa cells wasevaluated to screen for biocompatibility in vitro. The modifiedalginates identified in Example 2 as capable of forming hydrogels wereloaded into wells of 96-well plates which were coated withpoly-L-lysine. Unmodified alginate and saline were also loaded intowells of 96-well plates which were coated with poly-L-lysine ascontrols. A 100 mM BaCl₂ crosslinking solution was dispensed in all ofthe wells of the 96-well plate. The excess crosslinker was thenaspirated. HeLa cells were seeded into the wells and incubated for 3days at 37° C. in a humidified chamber.

A cell viability assay using3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasperformed, in which the media was aspirated from all wells and 100 μl ofDMEM media without phenol red and 100 MTT (5 mg/ml) were added to all ofthe wells of the 96-well plate. The plate was incubated for 4 hours at37° C. in a humidified chamber. After incubation, 85 μl of solution wasaspirated and 100 μl DMSO was added. Purple formazan crystals formduring the assay in proportion to the number of viable HeLa cellspresent in each well. The contents of each well were pipetted up anddown to solubilize the formazan crystals prior to measurement. The platewas incubated at 37° C. for 10 minutes after which the bubbles fromagitation were removed. The plate was read using a UV/Vis plate readerat 540 nm with a reference at 700 nm. The viability was normalized tocells seeded in wells with no alginate.

The results of the cell viability are shown in FIG. 3, which plots theeffect of selected modified alginates on HeLa cell line viability ascompared to the positive control (no alginate). Alginate (Alg) has aviability of 53%. The assay identified modified alginate polymers whichdisplayed decreased cytotoxicity relative to unmodified alginate. Thesewere selected for further analysis.

Example 4: High Throughput In Vivo Screening of Modified Alginates toAssess Biocompatibility

Current biocompatibility analysis methods are slow and requirehistological analysis. In order to screen the large numbers of modifiedalginates prepared using the combinatorial synthetic methods describedherein, a high throughput in vivo biocompatibility assay was used toassess the relative biocompatibility of alginate polymers.

1. High Throughput In Vivo Screening Protocol

8-12 week old male SKH1 mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). The mice were maintained at the animalfacilities of Massachusetts Institute of Technology, accredited by theAmerican Association of Laboratory Animal care, and were housed understandard conditions with a 12-hour light/dark cycle. Both water and foodwere provided ad libitum.

Injections were performed in accordance with ISO 10993-6: 2001. Prior toinjection all materials were sterilized via 0.22 μm filtration. The micewere anesthetized via isoflurane inhalation at a concentration of 1-4%isoflurane/balance O₂ to minimize movement. Their backs were scrubbedwith 70% isopropyl alcohol and the animals were injected with modifiedalginates in an array format on the mouse's back for high-throughputscreening. Six injections were made in each mouse with one of theinjections being an unmodified alginate control. Injection volumes were100 μl.

On days 1, 3, 7, 14, 21, and 28 post injection, host cell activity inresponse to the implantation of modified alginates was imagedkinetically using fluorescent whole animal imaging. 24 hours before invivo fluorescence imaging, 2 nmol of ProSense-680 (VisEn Medical,Woburn, Mass., excitation wavelength 680±10 nm, emission 700±10 nm)dissolved in 150 μl sterile PBS was injected into the tail vein of eachmouse to image cathepsin activity.

In vivo fluorescence imaging was performed with an IVIS-Spectrummeasurement system (Xenogen, Hopkinton, Mass.). The animals weremaintained under inhaled anesthesia using 1-4% isoflurane in 100% oxygenat a flow rate of 2.5 L/min. A binning of 8×8 and a field of view of13.1 cm were used for imaging. Exposure time and f/stop the relativesize of the opening of the aperture—were optimized for each acquiredimage. Data were acquired and analyzed using the manufacturer'sproprietary Living Image 3.1 software. All images are presented influorescence efficiency, which is defined as the ratio of the collectedfluorescent intensity to an internal standard of incident intensity atthe selected imaging configuration. Regions of interest (ROIs) weredesignated around the site of each injection.

Biocompatibility of the materials was examined 14 days post injection.The fluorescence intensity measured at the implantation site of modifiedalginates was compared with the fluorescence intensity measured at theimplantation site of and unmodified alginates. Modified alginatesexhibiting a smaller fluorescence intensity at the implantation sitethan the fluorescence intensity measured at the implantation site of anunmodified alginates were characterized as biocompatible. Modifiedalginates exhibiting a greater fluorescence intensity at theimplantation site than the fluorescence intensity measured at theimplantation site of an unmodified alginates were characterized as notbiocompatible.

The in vivo stability of the alginate gels was assessed at 28 days postinjection. Modified alginates which remained at the site of injectionafter 28 days were characterized as capable of forming mechanicallystable hydrogels in vivo. Modified alginates which were not present atthe site of injection after 28 days were deemed to not capable offorming mechanically stable hydrogels in vivo, and were classified as‘failures’.

Modified alginates characterized as both biocompatible and capable offorming mechanically stable hydrogels in vivo were identified as ‘hits’,and selected for further study.

2. Validation of the High Throughput In Vivo Screening Protocol

In order to validate the high throughput in vivo screening assaydescribed above, modified alginates were subcutaneously injected intomice in an array format and crosslinked in situ as described above. Micewere imaged on days 1, 3, 7, 14, 21, and 28 post injection usingfluorescent whole animal imaging, and tissue samples were collectedafter imaging for histological analysis. To obtain tissue samples forhistological analysis, mice were euthanized via CO₂ asphyxiation and theinjected biomaterial and surrounding tissue were excised. The tissueswere then fixed in 10% formalin, embedded in paraffin, cut into 5 μmsections, and stained using hematoxylin and eosin (H&E) for histologicalanalysis by a board certified pathologist.

Fibrosis was rated on a scale where a zero involved no fibrosis, a oneindicated partial coverage with one to two layers of fibrosis, a two isdesignated a thicker fibrotic layer that nearly covered the implant, anda three denoted concentric fibrotic coverage of the polymer. Bothpolymorphonuclear (PMN) cells and macrophages were rated on a scalewhere no observed cells were indicated with a zero, scattered cellsscored a one, numerous cells clustering on the sides of the polymerscored a two, and numerous cells surrounding the material resulted in athree. Both the histological score and fluorescence response normalizedto alginate were examined for the whole library and materials thatoutperformed unmodified alginate were judged to be biocompatible. Thiscorresponds to a normalized fluorescent signal of <100% and a fibrosisscore of <3.

Data captured using whole animal imaging was demonstrated to displayedsimilar temporal trends in cellular recruitment of phagocytes to thebiomaterials compared to histological analysis. Accordingly, the highthroughput in vivo screening method described above was validated.

Example 5: In Vivo Screening of Modified Alginates to QuantifyBiocompatibility

8-12 week old male SKH1 mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). The mice were maintained at the animalfacilities of Massachusetts Institute of Technology, accredited by theAmerican Association of Laboratory Animal care, and were housed understandard conditions with a 12-hour light/dark cycle. Both water and foodwere provided ad libitum.

Injections were performed in accordance with ISO 10993-6: 2001. Prior toinjection all materials were sterilized via 0.22 μm filtration. The micewere anesthetized via isoflurane inhalation at a concentration of 1-4%isoflurane/balance O₂ to minimize movement. Their backs were scrubbedwith 70% isopropyl alcohol and the animals were injected with a modifiedalginate. The injection volume was 100 μl.

Cathepsin activity was measured 7 days post injection using an in vivofluorescence assay to quantify the foreign body response to the modifiedalginate. 24 hours before in vivo fluorescence imaging, 2 nmol ofProSense-680 (VisEn Medical, Woburn, Mass., excitation wavelength 680±10nm, emission 700±10 nm) dissolved in 150 μl sterile PBS was injectedinto the tail vein of each mouse to image cathepsin activity.

In vivo fluorescence imaging was performed with an IVIS-Spectrummeasurement system (Xenogen, Hopkinton, Mass.). The animals weremaintained under inhaled anesthesia using 1-4% isoflurane in 100% oxygenat a flow rate of 2.5 L/min. A binning of 8×8 and a field of view of13.1 cm were used for imaging. Exposure time and f/stop—the relativesize of the opening of the aperture—were optimized for each acquiredimage. Data were acquired and analyzed using the manufacturer'sproprietary Living Image 3.1 software. All images are presented influorescence efficiency, which is defined as the ratio of the collectedfluorescent intensity to an internal standard of incident intensity atthe selected imaging configuration. Regions of interest (ROIs) weredesignated around the site of each injection.

Fluorescence images were captured 7 days post-injection illustratingrelative cathepsin activity at the point of injection of selectedmodified alginates. The fluorescence intensity was measured andnormalized to the fluorescence response measured using unmodifiedalginate in order to quantify the biocompatibility of the modifiedalginates. The results obtained for selected modified alginates areincluded in FIG. 4.

Example 6: Treatment of Diabetes in STZ-Induced Diabetic Mice

The transplantation of biocompatible alginate-encapsulated beta cellsoffers potential as a treatment for diabetes. Pancreatic rat islet cellswere encapsulated using fourteen biocompatible modified alginatepolymers identified using the assays detailed above (includingPF_N287_B_B4, PF_N287_F2, PF_N287_G3, PF_N287_B3, PF_N287_B_B8,PF_N287_A4, PF_N287_B1, PF_N287_E3, PF_N263_C12, PF_N63_A12, PF_N287_E1,PF_N287_D3, PF_N263_A7, and PF_N263_C6). Alginate-encapsulated isletscapsules were fabricated from 750 μl of a 4% (w/v) solution of eachmodified alginate in deionized water containing suspended 1,000 isletssuspended using the Inotech encapsulator (Inotech) set to a voltage of1.05 kV, a vibration of 1225 Hz, and a flow rate of 10-25 ml/min with a300 μm nozzle. Alginate was crosslinked in a 20 mM BaCl₂ solution. Afterencapsulation, the capsules were washed twice with HEPES solution, fourtimes with Krebs solution, and twice with RPMI-1640 medium.

The encapsulated rat islet cells were transplanted into STZ induceddiabetic mice. Prior to transplantation, the mice were anesthetizedunder continuous flow of 1-4% isofluorane with oxygen at 0.5 L/min. Ashaver with size #40 clipper blade will be used to remove hair to revealan area of about 2 cm×2 cm on ventral midline of the animal abdomen. Theentire shaved area was aseptically prepared with a minimum of 3 cyclesof scrubbing with povidine, followed by rinsing with 70% alcohol. Afinal skin paint with povidine was also applied. The surgical site wasdraped with sterile disposable paper to exclude surrounding hair fromtouching the surgical site. A sharp surgical blade was used to cut a0.5-0.75 cm midline incision through the skin and the linea alba intothe abdomen. A sterile plastic pipette was used to transfer the alginatemicrocapsules into the peritoneal cavity. The abdominal muscle wasclosed by suturing with 5-0 Ethicon black silk or PDS-absorbable 5.0-6.0monofilament absorbable thread. The external skin layer was closed usingwound clips. These wound clips were removed 7-10d post-surgery aftercomplete healing was confirmed.

Blood glucose levels in the STZ induced diabetic mice were monitoreddaily for between 20 and 30 days post-transplantation using a drop ofblood obtained by scrubbing the tail with 70% isopropyl alcohol andmaking a superficial cut on the skin of the tail to produce a drop ofblood. Mice were restrained during sampling in a rotating tail injector.

The blood glucose levels in the STZ induced diabetic mice followingislet transplantation are shown in FIG. 5. The dashed black linerepresents normoglycemia in mice. Pancreatic rat islet cells wereencapsulated in modified alginates were able to reduce the blood glucoselevels in all cases, and in some cases, were even able to inducenormoglycemia.

Example 7. Particles Prepared from Mixture of Modified Alginate(s) andUnmodified Alginate

The growing recognition of the parameters driving fibrosis in vivo hasbeen applied to the analysis of the performance of modified alginates.Intraperitoneal (IP) implantation of modified alginate capsules revealedthat modified alginates may result in abnormally shaped capsules whencrosslinked using conditions defined for unmodified alginates. Theseabnormally shaped capsules can complicate implementation andinterpretation of modified alginate capsules implanted IP. In an effortto improve the capsule morphology, formulation methods for use withmodified alginate microparticles were developed where modified alginateswere blended with a small amount of high molecular weight alginate.Particles prepared from this mixture yielded particles with improvedmorphology and stability.

A 6% solution of modified alginate (w/w) was combined 1:1 by volume witha 1.15% solution of unmodified alginate (w/w). After mixing, capsulesare formed by following this solution through an electrostatic dropletgenerator, followed by crosslinking of the polymer in a 20 mM aqueousbarium chloride solution.

Particles prepared from modified alginate 263_A12 microparticlesformulated with barium and mannitol were compared to particles preparedfrom 263_A12 blended with a small amount of unmodified SLG100 alginate(16% by weight). The particles prepared from a mixture of modifiedalginate and unmodified alginate produced more homogenous microparticlepopulations. Quantitative fluorescence analysis with prosense at severaltime points with modified alginates blended with SLG100 was performed.The results are shown in FIG. 6. Several reformulated modified alginatesdisplayed less inflammatory response at day 7 compared to the controlalginate. Initial experiments with large capsules (1.5 mm diameter) showcomparably clean capsules after 2 weeks in the IP space ofimmunocompetent C57BL6 mice.

Data collected to date with these controlled capsules indicates thatreformulation and capsule morphology can have a significant effect oninflammation as measured by prosense. An improved inflammation responseis observed in some polymers (FIG. 6), while others are impactednegatively.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A modified alginate comprising one or more covalentlymodified monomers defined by Formula I

wherein, X is oxygen, sulfur, or NR; R₁ is hydrogen, or an organicgrouping containing any number of carbon atoms, preferably 1-30 carbonatoms, more preferably 1-20 carbon atoms, more preferably 1-14 carbonatoms, and optionally including one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats, representative R₁ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; Y₁ andY₂ independently are hydrogen or —PO(OR)₂; or Y₂ is absent, and Y₂,together with the two oxygen atoms to which Y₁ and Y₂ are attached forma cyclic structure as shown below

wherein n is an integer between 1 and 4; and R₂ and R₃ are,independently, hydrogen or an organic grouping containing any number ofcarbon atoms, preferably 1-30 carbon atoms, more preferably 1-20 carbonatoms, more preferably 1-14 carbon atoms, and optionally including oneor more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative Rgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and R is, independently for each occurrence, hydrogenor an organic grouping containing any number of carbon atoms, preferably1-30 carbon atoms, more preferably 1-20 carbon atoms, more preferably1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R groupings being alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group.
 2. The modified alginate of claim 1, wherein X isoxygen.
 3. The modified alginate of claim 1, wherein X is NR.
 4. Themodified alginate of claim 1, wherein the modified alginate is asingularly modified alginate polymer.
 5. The modified alginate of claim1, wherein the modified alginate is a multiply modified alginatepolymer.
 6. The modified alginate of claim 5, wherein the modifiedalginate is one of the following


7. The modified alginate of claim 1, wherein more than 15% of themonomers in modified alginate polymer are covalently modified monomers.8. The modified alginate of claim 1, where the modified alginate can beionically crosslinked using Ca²⁺, Ba²⁺, or Sr²⁺ to form a hydrogel. 9.The modified alginate of claim 8, where the hydrogel induces a lowerforeign body response than a hydrogel formed from an unmodified alginatepolymer.
 10. A biocompatible ionically crosslinkable chemically modifiedalginate which is prepared by chemical modification of highly purifiedalginate, and purified after chemical modification to remove anyunreacted or partially reacted contaminants present with the chemicallymodified alginate, where the purified chemically modified alginateinduces a lower foreign body response than the chemically modifiedalginate prior to purification.
 11. The alginate of claim 10, whereinthe purified chemically modified alginate has the formula:

wherein, X is oxygen, sulfur, or NR; R₁ is hydrogen, or an organicgrouping containing any number of carbon atoms, preferably 1-30 carbonatoms, more preferably 1-20 carbon atoms, more preferably 1-14 carbonatoms, and optionally including one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats, representative R₁ groupings being alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy,aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; Y₁ andY₂ independently are hydrogen or —PO(OR)₂; or Y₂ is absent, and Y₂,together with the two oxygen atoms to which Y₁ and Y₂ are attached forma cyclic structure as shown below

wherein n is an integer between 1 and 4; and R₂ and R₃ are,independently, hydrogen or an organic grouping containing any number ofcarbon atoms, preferably 1-30 carbon atoms, more preferably 1-20 carbonatoms, more preferably 1-14 carbon atoms, and optionally including oneor more heteroatoms such as oxygen, sulfur, or nitrogen grouping inlinear, branched, or cyclic structural formats, representative Rgroupings being alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substitutedaroxy, alkylthio, substituted alkylthio, phenylthio, substitutedphenylthio, arylthio, substituted arylthio, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic,aminoacid, poly(ethylene glycol), peptide, or polypeptide group; or R₂and R₃, together with the carbon atom to which they are attached, form a3- to 8-membered unsubstituted or substituted carbocyclic orheterocyclic ring; and R is, independently for each occurrence, hydrogenor an organic grouping containing any number of carbon atoms, preferably1-30 carbon atoms, more preferably 1-20 carbon atoms, more preferably1-14 carbon atoms, and optionally including one or more heteroatoms suchas oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclicstructural formats, representative R groupings being alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, polyaryl, substitutedpolyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic, heterocyclic,substituted heterocyclic, aminoacid, poly(ethylene glycol), peptide, orpolypeptide group.
 12. The alginate of claim 10, wherein the modifiedalginate is combined with an unmodified alginate.
 13. The alginate ofclaim 12, wherein the modified alginate is defined by claim
 1. 14. Thealginate of claim 13, wherein the modified alginate is defined by claim6.
 15. The alginate of claim 10, wherein the alginate induces a lowerforeign body response in an assay comprising: injecting the modifiedalginate into a laboratory animal; and measuring the cathepsin activityinduced by the modified alginate using fluorescent animal imaging.
 16. Amethod of treating a disease or disorder in a human or animal patient,comprising: implanting or transplanting into a human or animal patient abiological material encapsulated in an ionically crosslinkedbiocompatible modified alginate defined by claim
 1. 17. The method ofclaim 16, wherein the biological material is cells.
 18. The method ofclaim 17, wherein the disease or disorder is diabetes and the cells arepancreatic islet cells.
 19. The method of claim 16, wherein the modifiedalginate is further crosslinked covalently.
 20. The method of claim 16,wherein the modified alginate further comprises unmodified alginate. 21.The method of claim 16, wherein the biological materials is encapsulatedin a microcapsule comprising the modified alginate.
 22. A combinatorialmethod for the synthesis of modified alginate polymers, comprising:reacting an unmodified alginate polymer with between one and threecompounds in a combinatorial fashion to obtain modified alginatepolymers, wherein the compounds possess a functional group selected fromthe group consisting of an amine, an alcohol, a ketone, an alkyne, ahalogen, and an azide; and purifying the modified alginate polymers. 23.The method of claim 22, further comprising screening the modifiedalginate polymers for the ability to form hydrogels.
 24. The method ofclaim 22, further comprising screening the modified alginate polymersfor biocompatibility.
 25. The method of claim 22, wherein the compoundsare selected from the group consisting of


26. The method of claim 22, wherein the modified alginate polymers arepurified by dialysis.
 27. The method of claim 22, wherein the modifiedalginate polymers are purified by chromatography.
 28. The method ofclaim 22, wherein the compounds form covalent bonds to the unmodifiedalginate polymer.